Thirty-nine ejaculates from 8 Holstein bulls were collected. Straws of semen (n=750) were distributed among dairies in three states. Ten straws per ejaculate were sent to Louisiana State University (LSU) Dairy Improvement Center for conventional polymerase chain reaction (PCR) analysis. Spermatozoal DNA was extracted and PCR analysis was done using one primer set amplifying a single copy 125 base pair (bp) section of the Bos taurus factor IX (Christmas factor) precursor (found on the X chromosome) and another primer set amplifying a single copy section of the Bos taurus sex determining region Y protein (SRY) gene (found on the Y chromosome). A 294 bp product from the Bos taurus glyceraldehydes-phosphate-dehydrogenase (GAPDH) was amplified as an internal control. Standard curves were designed using PCR products in known ratios. Gel electrophoresis and image analysis allowed for determination of predicted % Y chromosome-bearing spermatozoa (predicted % Y spermatozoa). Calf sex was reported and % male calves was determined between bull, ejaculate within bull, state, and location within state. Predicted % Y spermatozoa and % male calves showed significant correlation to each other. No significant variance between bull was found in predicted % Y spermatozoa or % male calves, but significant variance was found between ejaculate within bull for both. PCR technology used for determining the % Y spermatozoa in ejaculates was shown to be an adequate method to determine semen sex ratio.